Object: The objective of the proposal is to define counts which closely follow the binding of antigen to the specific receptors on the lymphocyte surface and relate them to the antigen-binding cell's maturation and eventual role in the humoral immune response. The population of mouse spleen cells which binds sheep erythrocytes has been selected for an ongoing multifaceted investigation of the immunologic role of the antigen-binding cell. We have developed physical methods for purifying antigen binding cells at various stages of the immune response which enable use to study some biochemical parameters of activation such as lipid turnover surface markers and immunologic potential in vitro or after adoptive transfer. Having succeeded in generating antigen-binding cells by primary exposure to antigen in vitro, we will determine whether this process requires cell division or new gene expression by means of inhibitors, and define whether cell-cell interactions are necessary. Means of stopping the response after expansion of antigen-binding cells but before terminal differentiation by temperature and inhibitors will be explored. Three hypotheses to explain the loss of receptor trypsin sensitivity after immunization will be tested. Antigen-induced receptor movements provide a visible sign that a signal has been generated by the crosslinking of membrane molecules, and the relationship of this signal to activation is being investigated. By sequential exposure to two reversible inhibitors of capping, we intend to determine the order of inhibitable events leading to capping. Single cell experiments are proposed to see whether capping cells are predisposed toward or away from terminal differentiation. Having demonstrated a failure to cap or regenerate receptors in TNP-tolerant B cells, we will extend the observation to other tolerance systems and study the impact of interactions with suppressor T cells and macrophages on these B cell membrane functions.